Post‐mortem investigation into a death involving doping agents: The case of a body builder

Abstract Introduction A young male was found dead on the bed of a hotel room. He was expected to take part in a bodybuilding competition the day after. During the site inspection, drugs of different types were found. The next day, an autopsy was performed. The evidence of cardiomegaly with organ congestion involving lung, liver, kidneys, adrenal glands, spleen and brain was confirmed by both the autoptic and the histopathological exam. However, the cause of death needed to be investigated. Methods A thorough toxicological investigation was undertaken by gas chromatography–mass spectrometry (GC‐MS), liquid chromatography‐high resolution mass spectrometry (LC‐HRMS) and liquid chromatography–tandem mass spectrometry (UPLC‐MS/MS) on samples of urine, blood and hair. Results and discussion Clenbuterol, a long‐acting selective beta2 agonist, was found in both blood (1 ng/ml) and urine (1 ng/ml), and evidence of its use was provided by the analysis of the 3‐cm hair (25 pg/mg). The main metabolite of drostanolone (2 alpha‐methyl‐androsterone), an anabolic steroid, was found in the urine (202 ng/ml), where an increased ratio of testosterone/epitestosterone (T/E = 11) emerged. Due to the results of the hair analysis, a long‐term use of various anabolic steroids was supposed. The integrated analysis of the results and the absence of other possible causes (such as trauma or cardiac conduction anomalies) led to the identification of the abuse of doping substances as the underlying cause of death. Conclusion Hair analysis has proven to be crucial in identifying drug misuse and the contributing cause of death.

(2 alpha-methyl-androsterone), an anabolic steroid, was found in the urine (202 ng/ml), where an increased ratio of testosterone/epitestosterone (T/E = 11) emerged. Due to the results of the hair analysis, a long-term use of various anabolic steroids was supposed. The integrated analysis of the results and the absence of other possible causes (such as trauma or cardiac conduction anomalies) led to the identification of the abuse of doping substances as the underlying cause of death.
Conclusion: Hair analysis has proven to be crucial in identifying drug misuse and the contributing cause of death.
Clenbuterol can be classified as a long-acting selective beta 2 agonist 1 that maintains its main therapeutic indication in the treatment of bronchial asthma. 2 Its established effects on the relaxation of bronchial smooth muscle, the decrease of mucus production and the increase of mucociliary transport in the airways support its clinical use. Further studies have shown its antiallergic activity against anaphylactic-induced asthmatic attacks in its capacity to stabilise the membrane of mast cells and basophils. In Italy, clenbuterol is available in the form of syrup or in tablets dosed at 20 μg, with the maximum recommended daily dose fixed at 40 μg. 3 In other countries, injectable solutions and aerosols are available.
The wide category of AAS includes more than 100 active principles, most of which lack governmental regulatory health authorities' approval for human therapeutic use (e.g., discontinued drugs, designer drugs and substances approved only for veterinary use), and only a few of which have therapeutic indications in animals (stanozolol) or in humans (e.g., testosterone), including hypogonadism, hormone sensitive neoplasms, medullary aplasia and osteoporosis. 4 Due to their impact on protein synthesis, 5 muscle growth and erythropoiesis, these substances have shown benefits in a variety of conditions, including HIV-related muscle wasting, muscle dystrophies, severe burn injuries, bone marrow failure, hereditary angioedema and growth retardation in children. 4 The anabolic-androgenic effects, which can be identified in the increase of mean muscle mass 6 and enhanced erythropoiesis, the stimulation of aggression and the decrease of the training-induced fatigue, sustain their widespread use among bodybuilders and athletes. The amplification of therapeutic effects, due to long-term use, leads to toxicity in term of high blood pressure with hypertrophy and coronary disease, liver damage, increased risk of prostate cancer, decreased libido, dermatological changes and metabolic imbalances.
A variety of side effects can occur when anabolic steroids are misused, ranging from mild effects to ones that are harmful or even life-threatening. Most are reversible if the user stops taking the drugs.
However, others may be permanent or semipermanent. 7 The main side effects on the human body regard the cardiovascular system (increase of blood pressure, blood clots, heart attack and stroke), 8 the hormonal system (decreased sperm production, testicular cancer and excessive body hair growth), the liver (peliosis hepatis, and tumours), the musculoskeletal system (tendon injury and short stature), the skin (acne and cysts) and the immune system (hepatitis and HIV). Most data on the long-term effects of anabolic steroids in humans come from case reports rather than from formal epidemiological studies.
Serious and life-threatening adverse effects may be underreported, especially since they may occur many years later. One review found 19 deaths in published case reports related to anabolic steroid use between 1990 and 2012; however, many steroid users also used other drugs, making it difficult to show that the anabolic steroid use caused these deaths. One animal study found that exposing male mice to steroid doses comparable to those taken by human athletes for one fifth of their lifespan caused a high frequency of early deaths. 7 The authors present here a fatal case of a bodybuilder involving the use of clenbuterol and AAS.

| CASE REPORT
The day before a bodybuilding regional competition, a 24-year-old male was found inanimate by his girlfriend, with whom he shared the room. She reported that they had spent the night before in a restaurant drinking some alcohol and went to sleep at 1:00 a.m. He went to the toilet only once during the night. At 6.00 a.m., she found the corpse of her boyfriend laying on the bed next to her. According to her, he habitually used thyroid medications and diuretics for sport activities, but he had not assumed any drugs before bedtime. 'As far as I know', she said, 'he didn't suffer from diseases of any type'.
Police inspection of the room revealed the presence of many drugs, one package of each, later identified as Diuresix ® (torasemide) ® , Monores ® (clenbuterol hydrochloride), Eutirox ® (levothyroxine sodium), Halotestin ® (fluoxymesterone) and Lasix ® (furosemide). Two empty disposable syringes were collected as well. The autopsy was carried out by the judicial authority the day after at the Pathological Anatomy Unit of Padua University, where samples were collected for toxicological ascertainments. Generalised organ congestion, including of the meningeal veins, spleen and the liver itself, was revealed by the macroscopic and the microscopic analysis. The lungs were affected by a massive edema with haemorrhagic extravasation, while the heart was characterised by a cardiomegaly with nonobstructive concentric intimal hyperplasia. Femoral and cardiac blood, urine, bile, gastric contents, vitreous humour and hair as well as fragments of kidney, brain and liver were collected for toxicology investigations.

| Toxicological initial screening
The case specimens were subjected to a laboratory post-mortem toxicological screening battery. Ethanol and volatiles were tested in blood and urine by a head space gas chromatography-flame ionisation detector on an Agilent 6890 system using an in-house validated procedure. An immunoassay test for pharmaceuticals and abused drugs was carried out on blood and urine using Siemens kits and following the recommendations of the manufacturer. A general unknown screening was performed on blood and urine by means of LC-HRMS on an Orbitrap LTQ (Thermofisher), and a target screening was performed by UPLC-MS/MS on a Xevo TQS (Waters). Subsequently, the quantification of clenbuterol in the blood was realised by a multiple reaction monitoring method using UPLC/MS-MS; clenbuterol and AAS steroids in the hair were identified and quantified by LC-HRMS, while clenbuterol and AAS in the urine were determined by GC-MS.
Diuretics were not detected in the urine or blood.

| Urine/blood sample-preparation for steroids
The urine/blood sample preparation for steroids consists of different phases: solid phase extraction (SPE) on C18 column, enzymatic hydrolysis, liquid-liquid extraction, final derivatisation and analysis with GC-MS. The SPE column, previously activated with water and methanol, was added with 5 ml of urine mixed with methyltestosterone and 19-noretiocolanolone-d4. After water washing, the elution was performed with methanol; the eluate was dried under nitrogen current, added with phosphate buffer (pH = 7) and hydrolysed with beta-Glucuronidase deriving from Escherichiacoli at 50 C for 2 h. The mixture was cooled and was added with carbonate/bicarbonate buffer (1:10) in order to obtain a solution adjusted at pH of 8.5-9. Finally, extraction was performed with 5 ml anhydrous pentane. The organic phase was dried and derivatised with a mixture of trimethylsilyltrifluoroacetamide/dithioerythritol/ammonium iodide (MSTFA/DTE/ NH 4 I) (1 ml:6 mg:4 mg) at 75 C for 20 min. The acquisition method was multiple ion monitoring, as shown in the supporting information Table S1.

| RESULTS AND DISCUSSION
Ethanol and other volatile substances tested negative in blood, while in urine, a low concentration of ethanol was revealed (0.17 g/L). The results concerning anabolising agents are reported in Tables 1-3. The extremely elevated T/E ratio can be considered as atypical,

T A B L E 1 Analytes concentrations (ng/ml) detected in blood and in urine
warranting follow-up investigations as to testosterone misuse in the living and indicating testosterone misuse in the deceased. 9 The presence of the testosterone esters in the hair suggests that the alteration of the ratio is due to exogenous drugs. The results arising from the blood and urine indicate a recent intake of clenbuterol that, due to its pharmacokinetic properties (t-max within 2. and overdosed use of the substance. In that case, the combination of clenbuterol with the anabolic stanzolol, found in the hair only, was considered synergistic and prodromic to severe cardiac toxicity, with a parallelism between long-term use documented through hair analysis and heart disease. Therefore, the cause of death was identified in the combined use of clenbuterol with an anabolic androgenic steroid that led to myocardial infarction even in the absence of underlying heart disease. 16 The presence of an underlying cardiac pathological alteration may be present in the form of dilatative cardiomyopathy, acute liver disease and severe coronary artery disease. 17,18 Furthermore, in an inclusive retrospective study 19 of 545 long-time anabolic steroid users, the incidence of atrial fibrillation was three times higher than in the control group, and the risk of thromboembolism was increased fivefold. Therefore, the consumers of AAS have an increased risk of hospital admission than their nonusing peers, with an overall mortality rate 0.7% higher than the controls and a risk of death People who misuse steroids can also typically 'stack' the drugs, taking two or more different anabolic steroids, mixing oral and/or injectable types and even taking compounds that are designed for veterinary use (e.g., stanozolol). They believe that different steroids interact to produce an effect on muscle size greater than the effects of each drug individually; however, this theory has not been proved scientifically.
Another common mode of steroid misuse is referred to as 'pyramiding', which typically involves taking a drug in a cycle of 6 to 12 weeks, then reducing its use gradually rather than starting and concluding a cycle abruptly. This is sometimes followed by a second cycle in which the person continues to train without using drugs. Steroid users believe that the pyramid system allows the body to adapt to high doses and that the drug-free cycle leads to the restoration of the hormonal system. It is interesting to report the existence of a technique called 'plateauing', which consists of the integrated or exclusive use of other steroids to avoid the onset of tolerance. However, the effects of pyramiding, cycling and plateauing have not been scientifically proven yet. 20

| CONCLUSIONS
This fatal case demonstrates that the inappropriate use of anabolising agents has become increasingly widespread in the bodybuilding community. Considering that an acute ingestion/injection rarely leads to poisoning or death, in most of cases, it is long-term abuse that causes death through cardiac damage or cancer. In this case, the concentration of clenbuterol in the blood and of drostanolone in the urine demonstrates a recent administration, with the achievement of toxic levels of clenbuterol in blood that, in association with a repeated use of multiple other AAS documented by hair results, led to death through cardiac, hepatic and metabolic toxicity. Indeed, the macroscopic and the microscopic analysis revealed multi-organ congestion involving veins and abdominal organs such the liver and spleen. Furthermore, from the same investigation emerged a massive pulmonary edema with haemorrhagic extravasation and a cardiomegaly with nonobstructive concentric intimal hyperplasia. The results from the hair analysis, considering the ways of incorporating the active principle from the blood to the keratin matrix, the hair growth rate, the window of detectability and the concentrations reported in the literature, support the diagnosis of long-term abuse of anabolising agents. The lack of a traumatic agent or of other diseases able to explain the death is useful to demonstrate that the only reasonable cause is the one dis-